The Ovistatic Effect of Saprotrophic Soil Fungi on Ascaris suum Eggs

The aim of the study was to evaluate the potential use of selected species of soil fungi (Fusarium oxysporum, F. sulphureum, F. verticillioides, and Penicillium expansum) for the bioregulation of the dispersive stages of a parasitic nematode – the large roundworm of pig (Ascaris suum). Experimental cultures containing A. suum eggs with soil fungi and control cultures without fungi were incubated at 26°C for 28 days. Microscopic observations of the developmental stages of the A. suum eggs (zygote, 2-8 blastomeres, morula/blastula, gastrula, and larva) were performed at 7, 14, 21, and 28 days. The API-ZYM test was used to semi-quantitatively determine the activity of 19 hydrolytic fungal enzymes. The cytotoxicity of the fungi was determined with a tetrazole salt MTT assay. Microscopic observations of A. suum eggs incubated in the presence of fungi up to day 28 did not show any signs of destruction to egg shells and/or penetration of the fungi into the eggs. The ovistatic effect of all tested fungi (F. sulphureum, P. expansum, F. verticillioides, and F. oxysporum; p<0.05) was seen only on the 7th day of incubation, whereas on the 14th day, only F. verticillioides and F. oxysporum showed an inhibitory effect on the embryogenesis of A. suum, and by the 28th day, only P. expansum. The API-ZYM test showed differences in the hydrolytic activity of the tested strains, while the MTT assay showed the high cytotoxicity of F. sulphureum, the moderate cytotoxicity of F. verticillioides and P. expansum, and the low cytotoxicity of F. oxysporum. Among the fungal strains studied, F. sulphureum showed the highest ovistatic effect, which may be related to its enzymatic activity and cytotoxicity.

Microbes, including fungi, constitute the main component of soil biomass, and the amount of carbon that fungi accumulate is equal to or exceeds the amount of carbon in plant roots (RITZ & YOUNG 2004). The importance of fungi in soil is also related to their diverse and complex interactions with other components of the soil environment. From an ecological point of view, the role of soil fungi as saprophytes participating in the decomposition of dead organic matter is particularly valuable. Additionally, some fungi species are a natural control for plant pathogens, and researchers are investigating those that could be used in biological plant protection (MOOSAVI & ZARE 2012;JYOTI & SINGH 2016). One important avenue in this field is the search for soil fungi which exert inhibitory effects on populations of parasitic nematodes of humans and animals, as soil infestation by the dispersive stages of intestinal parasites is a serious epidemiological problem in many regions of the world.
Nematodes A. lumbricoides and A. suum are the most common intestinal parasites of humans and pigs. It is estimated that infection by A. lumbricoides affects more than 1 billion people and causes 20,000 deaths annually (HOPKINS 1992). The reproductive potential of female A. lumbricoides is very high, as evidenced by the fact that one female can lay up to 250 thousand eggs per day. Eggs of Ascaris sp. show high resistance to most methods of inactivation (BROWNELL & NELSON 2006). Ascaris egg shells are composed of four layers: the inner lipoprotein layer (ascaroside layer), the chitin/protein layer, the lipoprotein vitelline layer, and the outer acid mucopolysaccharide/protein uterine layer. The ascaroside layer, which is responsible for the impermeability of the shell, consists of a mixture of 25% protein and 75% lipid-containing ascarosides. The chitinous layer contains chitin spindles in a protein matrix, and both the vitelline and uterine layers contain protein (WHARTON 1980;BROWNELL & NELSON 2006).
Particularly interesting are studies on the nematostatic activity of Fusarium and Penicillium sp. due to their hydrolytic activity (STÖCKLEIN et al. 1993;RAPP 1995;PEKKARINEN et al. 2000;DAHOT 2001;BARATA et al. 2002;FACCHINI et al. 2015) and cytotoxicity (CETIN & BULLERMAN 2005;TWARU¯EK et al. 2018). Studies regarding the influence of Fusarium and Penicillium sp. on the development of A. suum eggs are rather scarce and therefore, the aim of this study was to determine the effect of selected common saprophytic soil fungi on the development of A. suum eggs, particularly, in relation to their cytotoxicity and enzymatic activity.

Fungi
Strains of 4 species of saprophytic fungi obtained from soil were selected for the study: Fusarium oxysporum Schltdl. (CCM F-358, isolated from rice field soil, Vietnam), Fusarium sulphureum Schltdl. (KH 1808, isolated from an arable field, Poland), Fusarium verticillioides (Sacc.) Kuntze = Gibberella fujikuroi (Sawada) Wollenw. (KH 230, isolated from an arable field, Poland), and Penicillium expansum Link (CCM F-576, isolated from orchard soil, Poland). Fungal nomenclature is based on the Index Fungorum (http://www.indexfungorum.org). Single-spore cultures of the strains were grown on PDA medium (Merck). Discs (ø 4 mm) were cut out of the 3-week old mycelium from these cultures and placed on PDA medium. Incubation was carried out in the dark at 25°C for 21 days and then new discs (ø 4 mm) were cut and transferred to Petri dishes (ø 50 mm) containing a suspension of A. suum eggs.

Ascaris suum eggs
Fertilized eggs of A. suum were obtained from the uteri of female nematodes (n=60) derived from the intestines of pigs from organic (n=30) as well as traditional farms (n=30). The obtained eggs (2 ml), from organic and traditional farms, respectively, were mixed and centrifuged in distilled water (3x1000 rpm) for 3 minutes. Two ml of A. suum eggs were mixed with 48 ml of distilled water containing 0.05% formalin, 0.05% streptomycin sulphate, and 0.01% chloramphenicol (ARAUJO et al. 1995;BLASZKOWSKA et al. 2013). 50 ml of A. suum egg suspension was divided into 5 parts (10 ml each). A control culture (without fungi) and 4 experimental cultures (with fungi), containing 10 ml suspension of A. suum eggs each, were incubated in Petri dishes (ø 50 mm) at 26°C, 80% RH for 28 days.
Every week (on day 7, 14, 21 and 28 of incubation), 0.1 ml of A. suum egg suspension was collected from the control and experimental cultures and microscopic observations of embryonic development were carried out (Olympus CX21, Japan; x10; 40).
In 100 randomly observed eggs, the following development stages were determined: zygote, 2-8 blastomers, morula/blastula, gastrula, and larva. Microscopic observations of 100 randomly collected eggs from the control and experimental cultures were carried out 3 times (3 x 100 eggs).

Cytotoxicity of fungi
Cytotoxicity was determined by using tetrazole salt MTT (3[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide, Sigma Aldrich). The study was performed with the use of the swine kidney cell line (SK). The MTT colorimetric assay for cytotoxicity is based on the transformation of yellow-coloured tetrazole salts into violetcoloured, water-insoluble formazan crystals. The salt reduction takes place exclusively in the mitochondria of live and metabolically active cells. If cells are damaged or destroyed by the toxin, the salt will not transform and the yellow colour of the tetrazole salts will be retained (HANELT et al. 1994).
The cells were cultured in a medium supplemented with an antibiotic solution (penicillin and streptomycin, Sigma Aldrich) and fetal calf serum (Sigma Aldrich) in a CO 2 Hera Cell incubator (Heraeus, Germany) (5% CO 2 , 37°C, 98% humidity). The number of swine kidney cells was 2.2 × 10 5 /ml. Extracts were prepared from the strains of fungi grown in the Petri dishes (PDA medium). The strains of fungi were transferred to a sterile Stomacher bag and extracted twice with 50 ml of chloroform in laboratory paddle blender for 4 min (BagMixer® 400, Intersciences). The chloroform was then poured through a fluted filter into a round bottom flask and evaporated to dryness in a vacuum evaporator. Next, the evaporation residue was dissolved into 2 ml of chloroform using an ultrasound bath and pipetted into vials. Lastly, the extracts were evaporated under a stream of nitrogen. All extraction procedures were done in a chemical fume hood.
The MTT cytotoxicity assay was performed on microtitration plates. The control (agar medium without mycelium inoculum) and experimental cultures with soil fungi on PDA were diluted in MEM (Minimum Essential Medium Eagle, Sigma Aldrich) medium containing 1.7% ethanol (POCH) and 0.3% DMSO (Merck) as 1:2. Consecutive dilutions were transferred onto MTT assay plates containing SK cells suspended in medium with ethanol, DMSO, and 10% FCS (fetal calf serum, Sigma Aldrich).
During the next 48 hrs, the plates were in a CO 2 Hera Cell incubator (Heraeus, Germany) (5% CO 2 , 37°C, 98% humidity). Next, 20 µl MTT solution in PBS (Merck) was added to the wells, and the whole set was incubated for another 4 hrs. Following liquid removal from the wells, 100 µl DMSO was added as a solvent for the formazan crystals. After 5 min of shaking (shaker Titramax 101, Heidolph), cytotoxicity was quantified with a micro-plate spectrophotometer (Ledetect 96 Microplate Reader, Labexim Products, Lengau, Austria) coupled with MikroWin 2010 OEM version (Mikrotek Laborsysteme GmbH, Overath, Germany), based on absorbance measured at 510 nm wavelength, which corresponded to maximum absorption of the formazan derivative. In the case of absorbance lower than 50% of the cell division activity, all analyzed samples were considered toxic.
Cytotoxicity IC 50 (sample concentration at which cell proliferation is inhibited by 50% compared to control cells) was determined based on consecutive dilutions according to the scale of HANELT et al. (1994) (Table 1). The cytotoxicity of the analyzed fungal strains was reduced by the IC 50 value of the control.

Statistics
Statistical analyses were performed using Statistica version 12 software (StatSoft. Inc). In order to determine differences between the two groups (control vs experimental), the t-test for independent samples was applied. Differences with p<0.05 were considered significant.

The influence of soil fungi on the embryonic development of Ascaris suum
The presence of mycelium of the tested fungal species in the experimental cultures of A. suum eggs influenced the course of embryogenesis in different ways (Fig. 1).
On the 7th day of incubation, all examined species of fungi demonstrated an inhibitory effect on the embryogenesis of A. suum. The inhibitory effect on the development of A. suum eggs (expressed as a higher percentage of eggs in the zygote stage, compared to the control, p<0.05) was observed in the following fungal species: F. sulphureum, P. expansum, F. verticillioides, and F. oxysporum (in order from the strongest to the weakest inhibitory effect). Simultaneously, a significantly lower percentage of A. suum eggs at the morula/blastula stage (p<0.05) was found in cultures incubated with fungi in comparison to control culture.
On the 14th day of embryogenesis, a significantly higher percentage of A. suum eggs in the zygote stage was found only in cultures containing F. oxysporum compared to the control culture. A higher percentage of eggs in the morula/blastula stage were found in cultures incubated with F. verticillioides and a lower percentage in those incubated with F. oxysporum (p<0.05). In the same period, a significantly lower percentage of eggs in the larval stage was found only in A. suum cultures incubated with F. verticillioides.
On the 21st day of incubation, the percentages of particular developmental stages of A. suum eggs did not show statistically significant differences between control and experimental variants.
On the 28th day of incubation, the highest percentage of eggs in the zygote stage with the lowest percentage of eggs in the larval stage was found in cultures containing P. expansum. A higher percentage of eggs in the morula/blastula stage (p<0.05) compared to the control culture was found only in the presence of F. verticillioides (Fig. 1). The enzymatic activity of fungi In the API-ZYM test (Table 2), the activity of peptide hydrolases (leucine arylamidase, valine arylamidase) was low/moderate (1-3) or undetectable (cystine arylamidase, trypsin, chympotrypsin). Low activity of leucine arylamidase was observed in F. oxysporum and F. sulphureum, while moderate activity was observed in P. expansum (3). Low activity of valine arylamidase was observed only in F. sulphureum, while none of the strains gave a positive reaction for cystine arylamidase.
In all fungi, activity of esterase (C4) and lipase esterase (C8) was observed, while none of the strains tested produced lipase (C14).
High acid phosphatase activity was observed in F. oxysporum, F. sulphureum, and P. expansum and low activity was seen in F. verticillioides. High activity of naphthol-AS-BI-phosphohydrolase was observed in F. oxysporum, F. sulphureum, and P. expansum, and low activity was found in F. verticillioides. á-galactosidase activity was demonstrated in F. sulphureum and P. expansum, whereas â-galactosidase was produced only by P. expansum. No activity of â-glucuronidase or á-fucosidase was observed in any of the strains studied. Low activity of á-mannosidase was observed in F. sulphureum and P. expansum.

Cytotoxicity of soil fungi in MTT test
The MTT test performed on the selected fungal strains showed various levels of cytotoxicity: high (IC 50 0.015 cm 2 /ml) for F. sulphureum, moderate for F. verticillioides and P. expansum (IC 50 0.488 cm 2 /ml and IC 50 3.906 cm 2 /ml respectively), and low (IC 50 31.25 cm 2 /ml) for F. oxysporum (Table 3). Table 2 The production of 19 hydrolases by soil fungal species in the API-ZYM test

Discussion
The ovistatic effect of the tested fungal strains (demonstrated as a delayed initiation of zygote division and reaching of the morula/blastula stage) manifested itself mainly on the 7th day of incubation for all tested fungi: F. sulphureum, P. expansum, F. verticillioides, and F. oxysporum, and corresponded with their cytotoxicity in the MTT assay (high -F. sulphureum, moderate -P. expansum and F. verticillioides, and low -F. oxysporum). None of the tested strains showed an ovicidal effect on the eggs of A. suum. Microscopic observations of A. suum eggs incubated in the presence of fungi up to the 28th day of development did not reveal any symptoms of destruction of egg shells and/or penetration of the fungi into the eggs. This is also confirmed by the results of BLASZKOWSKA et al. (2013) under similar conditions, where hyphal penetration by F. oxysporum and P. expansum into the A. suum eggs was observed in 10-20% and 10-15% of eggs, respectively, only after 28, 48, and 60 days of incubation. In our study, only F. verticillioides and F. oxysporum at day 14 and P. expansum at day 28 of incubation showed an inhibitory effect on the embryogenesis of A. suum. This may indicate a weakening of the antagonistic effect of the examined fungi during incubation, which is contrary to the results of BLASZKOWSKA et al. (2013), where this effect increased with the length of exposure (up to 60 days). Very high ovicidal activity of F. oxysporum on Toxocara canis eggs was demonstrated by CIARMELA et al. 2010. In our study, the highest ovistatic effect on A. suum up to day 7 was shown by F. sulphureum, which may be related to its high enzymatic activity and cytotoxicity. In a study by CIARMELA et al. 2010, F. sulphureum demonstrated an ovicidal effect on the eggs of T. canis. These differences seem to confirm that T. canis eggs are more sensitive to the antagonistic effects of fungal strains than A. suum eggs, as demonstrated by comparative studies in the presence of Fusarium culmorum, Metarhizium anisopliae, Paecilomyces fumosoroseus, Trichoderma viride, and Trichothecium roseum (MAZURKIEWICZ-ZAPA£OWICZ et al. 2014). These conflicting results in the studies of different authors concerning the same fungal species may be due to differences in the metabolic activity of different strains of a given species.
A surprising finding was the high percentage of A. suum eggs in the control culture (more than 50%) that remained in the zygote stage even on the 28th day of embryogenesis. The A. suum eggs used for the tests (both in experimental and control cultures) were obtained from 60 nematodes collected from pigs from organic (n=30) and traditional (n=30) farms. On organic farms, one of the methods of reducing parasitic infections is the use of feed additives in the form of medicinal plants containing active substances that reduce the level of parasitic infections in the host organism. These substances affect the physiological functions of the parasite, such as mobility, food absorption, or reproductive processes (MASAMHA et al. 2010). In our study, at the organic farm from which the A. suum were obtained, feed contained a mixture of herbs such as garlic (Allium sativum), pumpkin (Cucurbita pepo), thyme (Thymus vulgaris), mugwort (Artemisia vulgaris), and fennel (Foeniculum vulgare). These plant species show different levels of antagonistic activity against nematodes (COSTA et al. 2008;EL SHENAWY et al. 2008;BURKE et al. 2009;FEITOSA et al. 2013;GIARRATANA et al. 2014). The presence of these herbal species in the host's diet was most likely the cause of the inhibition of the embryonic development of A. suum (to the same degree in the zygote stage), both in the control and experimental cultures.
We observed no morphological damage to the A. suum eggs which might suggest a biochemical basis for the antagonistic effects of the tested fungal strains. Lipases and proteases produced by  (AHMAD et al. 2006). In our study, the delayed embryonic development of A. suum may also have been caused by the secretion of mycoenzymes. Among the tested peptide hydrolases (leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, and chymotrypsin), only leucine arylamidase showed weak activity in the test fungal strains except F. verticillioides. Valine arylamidase activity was detected only in F. sulphureum.
The API-ZYM test makes it possible to determine the activity of only a few select proteolytic enzymes, which in this study showed low or no activity. This observation does not exclude the participation of other proteolytic enzymes in the degradation of A. suum egg shells, as fungal species from genera Fusarium and Penicillium have been reported to exhibit proteolytic activity (BARATA et al. 2002;PEKKARINEN et al. 2000;DAHOT 2001). Compared to the low activity of arylamidases, we observed high activity of N-acetyl-â-glucosoaminidase (NAG), a high molecular-weight hydrolytic lysosomal enzyme which breaks chemical bonds in the glycosides and amino sugars that form structural components in many tissues. This enzyme is necessary for the degradation and disposal of various parts of the cell, including the cell membrane, and its high activity in this study suggests that it may be involved in A. suum egg degradation (PUSZTAHELYI & PÓCSI 2014). High activity of alkaline and acid phosphatases was also observed in the tested fungal strains, except in F. verticillioides. These enzymes are involved in catalyzing the dephosphorylation of various phosphate esters, which may suggest that, together with other enzymes, they may exert an inhibitory effect on the embryonic development of A. suum. LÝSEK and KRAJCI (1987) found that proteases and chitinases are involved in lysing the chitinprotein complex of the chitinous envelope layer of A. lumbricoides eggs. These enzymes, considering the high activity shown in the API ZYM test, may have participated in the ovistatic effect of the tested fungi on A. suum eggs, the shells of which are composed of chitin and a mixture of glycosides knowns as ascarosides. Exudation of some enzymes may cause degradation of diol ascarosides and acetylated aglycone that constitue 70% of ascarosides in the layers of A. suum egg shells, and thus increase their permeability (KU . The important role of mycoenzymes (lipases and proteases) in the destruction of eggs has been demonstrated previously in phytopathogenic nematodes whose egg shells are composed of protein, chitinous, and lipid layers (MOOSAVI & ZARE 2011). In our study, the lipolytic activity of the tested fungal strains may have caused changes in the permeability of the cell membranes of the developing embryos and interfered with the course of the embryogenesis of A. suum eggs.
Fungi are known to produce other metabolites such as allergens, mycotoxins, antibiotics, glucans, and volatile organic compounds (VOCs). In the present study all fungi belong to species known to produce toxic metabolites (WEIDENBÖRNER 2001). Many fungal strains produce mycotoxins, such as patulin produced by P. expansum (ANDERSEN et al. 2004), moniliformine by F. oxysporum, and fumonisin by F. verticillioides (COVARELLI et al. 2012). Some important secondary metabolites of the genus Penicillium are mycotoxins (such as patulin and griseofulvin) and antobiotics (PERRONE & SUSCA 2017), which may affect the growth and development of other organisms. A single fungus strain can produce several different mycotoxins and one mycotoxin can be produced by many different fungi.
Our results indicate that the degree of antagonistic interactions between soil fungi and A. suum eggs may depend on the metabolic specificity of strains within the species. Therefore, possible use of saprotrophic soil fungi in the reduction of dispersive stages of nematodes should be preceded by interdisciplinary studies of the biochemical and cytotoxic activity of individual strains.